The present invention relates generally to methods and devices of detecting proteins. Specifically, the invention is directed to methods and devices for detecting proteins at lower pH""s using various buffers.
Methods for the detection of urinary proteins are often more sensitive to albumin than to other urinary proteins. It is important, however, to detect proteins other than albumin, especially in the case of Bence-Jones proteinuria. The detection of proteins should cover a wide range of protein concentrations since the decision levels and recommended actions to be taken by clinicians will vary depending on the concentration of protein detected in a patient""s urine. For example, persistent proteinuria greater than 50 mg/dL represents strong evidence of renal disease whereas a protein level of greater than 300 mg/dL is consistent with a diagnosis of nephrotic syndrome. A concentration of protein in urine of greater than 800 mg/dL suggests massive protein loss and warrants a renal biopsy and/or steroid therapy. Accordingly, it is apparent that a test for protein in urine should be effective over a wide range of protein values.
Various methods for the determination of protein in aqueous fluid have been reported in the literature. These methods include the Kjeldahl method, biuret method, Lowery method, dyestuff combination method, UV method and fluorometric method. In general, proteins react with various substance including dyes such as bromphenol blue, coomassie brilliant blue and eosine, as well as metal ions such as silver (I), copper (II), zinc (II) and lead (II).
Urine contains amounts of salt that vary between individuals. Some of these salts may act as a buffer when a diagnostic assay demands operation at a pH different than that of the urine sample. For example, urine sample with salts such as bicarbonate, acetate or phosphate tend to resist the lowering of pH. Creatinine, a common component of urine, may also act as a buffer in resisting the lowering of pH. The result of this buffering is that some urine samples will cause a strip pH to be higher than optimal. A higher than optimal strip pH may result in color generation of the protein indicator dyes, providing a false indication of the presence of protein. It is desired to have a buffing system that reduces such urine effects, while not interacting with the chemistry occurring.
Accordingly, a need exists for a method and device for detecting proteins over a wide range of clinical concentrations and pH""s.
According to one embodiment, an assay for the determination of protein in an aqueous test fluid comprises combining the test fluid with a buffer and a dye. The buffer is selected from citrulline, malonic acid, cyanoacetic acid, citraconic acid, methyl phosphonic acid, sarcosine, saccharin, or combinations thereof. The buffer is added in sufficient quantity to maintain the pH of the assay including the test fluid at a selected target pH range within a range of from about 2.0 to about 3.0. The dye has a pKa which is enables it to operate as a protein indicator at the target pH range. The dye has an affinity for protein such that it will provide a detectable response in the presence of greater than about 15 mg/dL protein, to thereby render the assay suitable for the detection of total protein in the test fluid.
According to another embodiment, a dry device for use in determining protein levels in a fluid test sample comprises an absorbent material having absorbed therein a buffer and a dye. The buffer is selected from citrulline, malonic acid, cyanoacetic acid, citraconic acid, methyl phosphonic acid, sarcosine, saccharin, or combinations thereof. The buffer is added in sufficient quantity to maintain the pH of the assay including the test fluid at a selected target pH range within a range of from about 2.0 to about 3.0. The dye has a pKa which enables it to operate as a protein indicator at the target pH range. The dye has an affinity for protein such that it will provide a detectable response in the presence of greater than 15 mg/dL protein upon contact between the device and the fluid test sample, to thereby render the device suitable for the detection of total protein in the fluid test sample.
According to a further embodiment, an assay for the determination of protein in an aqueous test fluid comprises combining the test fluid with a buffer and a dye. The buffer is selected from citrulline, malonic acid, or combinations thereof The buffer is added in sufficient quantity to maintain the pH of the assay including the test fluid at a selected target pH range within a range of from about 2.0 to about 3.0. The dye has a pKa which enables it to operate as a protein indicator at the target pH range and which has an affinity for protein such that it will provide a detectable response in the presence of greater than about 15 mg/dL protein, to thereby render the assay suitable for the detection of total protein in the test fluid.
According to yet a further embodiment, a dry device for use in determining protein levels in a fluid test sample comprises an absorbent material having absorbed therein a buffer and a dye. The buffer is selected from citrulline, malonic acid or combinations thereof. The buffer is added in sufficient quantity to maintain the pH of the assay including the test fluid within a selected target pH range within a range of from about 2.0 to about 3.0. The dye has a pKa which enables it to operate as a protein indicator at the target pH range and which has affinity for protein such that it will provide a detectable response in the presence of greater than 15 mg/dL protein upon contact between the device and the fluid test sample, to thereby render the device suitable for the detection of total protein in the fluid test sample.